THE INTEREST in natural antimicrobial preservatives is rapidly grown due to their exceptional properties such as biodegradability, biocompatibility, and lower toxicity. On the contrary, common synthesized preservatives are more toxic, partially or non-degradable, and have undesirable side effects on human health and the environment. Furthermore, the resistance of microorganisms to current antimicrobial preservatives is increasing. In the present study, rhamnolipid was produced from Pseudomonas aeruginosa ATCC 9027, and twelve congeners of rhamnolipids were elucidated by ESI-MS. Additionally, rhamnolipids reduced the surface tension of water to 26mN/m with a critical micelle concentration value of 22mg/L. The produced rhamnolipids showed prominent lower toxicity against the brine shrimp Artemia salina than those of positive control and methylisothiazolinone. A significantant antimicrobial effect of rhamnolipids was achieved in vitro, whereas the disc diffusion assay showed noticeable inhibition zones around the paper discs saturated with different concentrations of the rhamnolipid produced in this study, with MICs ranged between 8 and 336μg/ml against Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans and Aspergillus brasiliensis. Moreover, a promising preservation efficacy of the produced rhamnolipid was obtained when applied to a personal care formulation model even after 28 days of incubation, with reduction values of about 3 and 1 expressed in log units for bacteria and fungi, respectively. However, rhamnolipids could be recommended as a powerful nontoxic bio-preservative for personal care formulations.